Recently, we have cloned and characterized NDRG4, a novel member of the NDRG family (N-myc downstream-regulated gene), from human, mouse and zebrafish and documented that this phylogenetically distinct member of the NDRG family is expressed exclusively in the heart and brain of both fish and mouse. NDRG4 is expressed predominantly in the embryonic and adult myocardium and although multiple isoforms of NDRG4 protein were detected in developing brains, only one isoform (NDRG4-S) was detected in the developing heart. Morpholino knockdown experiments in zebra fish resulted in dramatic thinning of the myocardium, decreased myocyte number, abnormal looping and cardiac failure in the embryo. In addition, mice heterozygous for a hypomorphic Ndrg4 allele show dramatic somatic and myocardial growth retardation shortly after birth. We hypothesize that NDRG4 plays a role in regulating cardiomyocyte proliferation during cardiac morphogenesis and plays an essential role in maintenance of the mature, differentiated myocyte phenotype in the adult. We therefore propose to 1) Define the role of ndrg4 in early heart development of zebrafish in vivo. Antisense morpholino oligonucleotides will be designed to the 5'-untranslated region of ndrg4 and injected into zebrafish embryos. In addition a reverse genetic TILLING screen will be used to obtain missense and nonsense mutations in Ndrg4. These approaches will be used to evaluate alterations in cardiac looping and chamber formation and determine if there are perturbations in the normal program of cardiomyocyte proliferation, apoptosis, and myocyte gene expression. 2) Delineate the role of Ndrg4 on murine cardiac development, in vitro. Mouse ES cells homozygous for a null mutation in Ndrg4 (Ndrg4 / ) will be used to determine the role of Ndrg4 in proliferation, cell cycle control, and sequential cardiomyocyte gene expression in the embryoid body model of myocyte differentiation utilizing immunofluorescent sorting of the myocyte population, qRT-PCR and FACs analysis of cycle regulation. A proteomic strategy employing LC-MS-MS will be used to identify NDRG4 associated proteins in order to place NDRG4 within the appropriate protein interaction networks for systems level analysis. 3) Determine the role of Ndrg4 in prenatal and postnatal cardiac development, in vivo. Animals homozygous for a conditional loxP Ndrg4 allele will be used in conjunction with myocardial specific expression of Cre recombinase under control of the rat troponin T promoter and the doxycyline inducible cTnT-nrtTA mouse line to determine the effects of tissue and temporal specific deletion of Ndrg4 in developing myocardium and adult mouse heart. Epistatic interactions between Ndrg4 and Ndrg2 will be analyzed by evaluation of embryos with compound heterozygous and homozygous null mutations in the Ndrg4 and Ndrg2 alleles. PUBLIC HEALTH RELEVANCE: Our preliminary data shows that NDRG4, a phylogenically distinct member of the NDRG family is expressed almost exclusively in the heart and brain of both zebra fish and mouse. Morpholino knockdown experiments in zebra fish result in dramatic thinning of the myocardium, decreased myocyte number, abnormal looping and cardiac failure in the embryo. Therefore, the goal of this project is to determine the role of this novel cytoplasmic protein in cardiac development and postnatal myocardial growth.